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Microalgae Culture Protocol

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Microalgae Culture Protocol t Microalgae Culture Protocol Aquaculture Division Harbor Branch OceanographicInstitutionInc. t MICROALGAE CULTURE PROTOCOL Aquaculture Division HARBOR BRANCH OCEANOGRAPHIC INSTITUTION INC. Juan C. Jaramillo Harbor Branch Oceanographic Inst. 5600 U.S. I North Flo Pierce, Florida 34946 Key words: Sterile techniques, microalgae, nutrients, growth. Abstract The protocol for culturing microalgae at the Aquaculture Division of Harbor Branch Oceanographic Institution is explained in detail. The purpose of this document is to serve as a step-by-step guide for technicians in the microalgae laboratory. Sterile techniques, culture vessel cleaning and preparation, nutrient and media preparation, inoculations, and culture room maintenance are described. Systems diagrams and flow charts indicating culture techniques and paths are included. Appendices on autoclaving, filtration and chemical reagents preparation are also included. © Copyright 1996. Harbor Branch Oceanographic Institution Inc. • Table of Contents Page Introduction 1 1.0 Stock Cultures (10 ml) 2 1.1 Clones Used at HBOI Aquaculture 2 1.1.1 Tiso (Isochrysis galbana) 3 1.1.2 Cg (Chaetoceros gracilis) 3 1.1.3 Tw iTh.alassiosira weissflogiiy 3 1.2 Culture Vessel Preparation 3 1.3 Media Preparation 4 1.4 Transfers 6 1.5 Stock Culture Table 7 2.0 Small-scale Cultures (500-6000 ml) 7 2.1 Culture Vessel Preparation 8 2.2 Culture Media Preparation 8 2.3 Inoculation 9 2.4 Small-scale Culture Table 10 3.0 Carboy Cultures (16-20 liters) 10 3.1 Culture Vessel Preparation 1 1 3.2 Culture Media Preparation 12 3.3 Inoculations 14 3.4 Culture Room 14 4.0 Cylinder Cultures in Room #2 (185- 208 liters) 16 4.1 Culture Vessel Preparation 17 4.2 Culture Media Preparation 1 8 4.3 Inoculation 19 4.4 Harvest 20 4.5 Culture Room 20 6.0 Algae Greenhouse Cultures 21 5.1 Culture Vessel Preparation 2 1 5.2 Culture Media Preparation 22 5.3 Inoculations 23 5.4 Greenhouses 23 Appendix Autoclaving 25 Appendix II Filtration and U.V. System 30 Appendix III Thiosulfate and Silicates 32 © Copyright 1996. Harbor Branch Oceanographic Institution Inc. Introduction At the Aquaculture Division only few species of microalgae are used to feed our organisms. The most important is the small flagellate Isochrysis g albana which is used to feed larval as well as juvenile and adult clams. The second most important species in our lab is Chaetoceros gracilis, a small diatom used mostly to feed juvenile and adult stages of clams and larval stages of shrimp and other invertebrates. Also used for the same purpose is the diatom Th alassiosira w eissflogii. In addition to these species, the lab cultures Ellipsodon sp., Tet raselmis sp., Nannocloropsis sp., and Chlorella sp., to feed clams, rotifers, artemia and ornamental larval shrimp. Other clones not mentioned here are maintained in our small collection but they are rarely used for feedings. Of utmost importance to the operation of growing microalgae in our laboratory is the proper utilization of Sterile Techniques in every step of the process in the first stages of batch cullure (inside laboratory, rooms #1 and #2). Without care taken in handling containers, media and instruments, you are likely to get contamination soon after you order new stock cultures. Those cultures are not axenic I to begin with (axenic cultures are a myth, really l), but they are pretty clean, and will last for years without a problem if precautions are taken. Some people prefer to move cultures fast through the laboratory to avoid "crashes">, I prefer to move slower, let things get dense enough, and be very meticulous with the technique to avoid those crashes. That way you are never in a rush and you do not have to live stressed and on "the edge" all the time. Our operation starts with 10 ml sock cultures that are progressively scaled up to about 3000 liters. This guide takes you from the very beginning of the process to the stage where algae is ready to be fed to the animals in our facility. 1 Cultures with only one species of any microorganism. If a culture has bacteria, but still survives well, it is called "monoalgal culture". 2 Term used when a culture abruptly dies due to contamination or nutrient depletion. Generally a crash involves decoloration of the culture, the presence of clumps and sometimes a foul smell. © Copyright 1996. Harbor Branch Oceanographic Institution Inc. 1 1.0 Stock Cultures (10 ml) Stock cultures are those used to start new batches of cultured algae for the laboratory. They are obtained by two different means. One is the use of a technique, called Single Cell Isolation from water samples collected in the field. This technique is time consuming, difficult to master and the results are unreliable, therefore, it is not been used at Harbor Branch. The best way to obtain good quality stock cultures is to buy them from other laboratories. We use two main sources: The University of Texas (UTEX), and Bigelow Laboratories (CCMP). The collection at UTEX is extensive and the clones obtained from them in the last years have been very reliable. They are also inexpensive ($10 per test tube). CCMP also has a good collection but the cultures are considerably more expensive ($40-$100 per test tube). Cultures can also be obtained from the American Type Culture Collection (see addresses below). 1.1 Clones Used at HBOI Aquaculture The clones used at HBOI can be ordered by letter, phone, fax, or usmg the internet at: Culture Collection of Algae (UTEX) Department of Botany The University of Texas at Austin Austin, Tx 78713-7460 Tel: (512) 471-4019 Fax: (512) 471-3878 URL: http://www .botany.utexas.edu/i nfores/utex/ National Center for Culture of Marine Phytoplankton (CCMP) Bigelow Laboratory for Ocean Sciences West Boothbay Harbor, Maine 04575 Tel: (207) 633-9600 Fax: (207) 633-9641 American Type Culture Collection 1230I Parkland Drive Rockville, Maryland 20852-1776 Tel: (800) 638-6597 Fax: (301) 231-5826 © Copyright 1996. Harbor Branch Oceanographic Institution Inc. 2 1.1.1 Tiso (Isochrysis galbana) We have one clone of Tiso from the UTEX collection. Its code is: LB2307 Size: 4-7 urn x 4 urn Doublings day-t: 2.89 TO Range: 16-34°C (28°C optimum) 1.1.2 Cg (Chaetoceros gracilis) We have one clone of Cg from the UTEX collection. Its code is: LB 2375 Size: 5-7 urn x 4 urn Doublings day-i: 4.3 TO Range: Not determined (28-32°C optimum) 1.1.3 Tw tTh.alassiosira weissflogii) We have one clone of Tw also, called 'Actin,' from The University of Miami. This clone can also be obtained from CCMP. Size: 12-24 um diameter Doublings day-r: 1.39 TO Range: IO-30°C (25-30°C optimum) When cultures are ordered make the people at Receiving aware of the shipment so they do not put them in the freezer or leave them exposed to the sun. Once in the lab, place them in a rack on the culture table and wait a day or two before you transfer them to new test tubes. 1.2 Culture Vessel Preparation Stock cultures come from the providers in glass or plastic test tubes (10 ml). They are transferred into borosilicate test tubes with lose caps or lids. Tubes must be washed even if they are just out of the box. To wash tubes follow the next steps: © Copyright 1996. Harbor Branch Oceanographic Institution Inc. 3 • submerge them in a bath of water with Alconox® detergent and leave them soaking for 24 hrs. • Rinse them very well with Reverse Osmosis (R.O.) water several times. The last rinse is done using distilled water or deionized water. It is very important that all traces of soap are removed from inside the tubes. • Pour 10 or 15 ml of D.l. or distilled water in each tube. This is done to trap copper residues left by the autoclaving process (The autoclave has copper plumbing) that can eventually kill the microalgae. • Autoclave tubes for 30 minutes at 125°C (see the "Autoclaving" appendix). • Let tubes cool down for 24 hr and store them In a dark, cool clean place until use. 1.3 Media Preparation We buy nutrient stocks ("algae food" or f/2) rather than prepare them from chemical reagents in our lab, mainly to save time. They are bought in 5 gallon containers and re-bottled in smaller teflon or plastic containers for (internal) indoor use. We order nutrients from: Aquacenter Tel: (800) 748-8921 Fax: (601) 378-2862/378-2861 There are two solutions to buy: Solution A (trace metals / catalog # 1081 OA) Solution B (P, N and vitamins / catalog # 1081OB) To sterilize media for the stock cultures, seawater must be passed through a series of filters ranging from 25 urn to 3 um, then through U.V., and finally through a fine 0.2 urn filter (Nalgenew, Fig 1.) © Copyright 1996. Harbor Branch Oceanographic Institution Inc. 4 Figure I. 0.2 urn filter To do this make sure the filtration system is working and the U.V. lights are turned on (see the "Filtration and U.V. System" appendix). Filter water into a clean 500 ml Pyrex® glass container with a screw cap (Fig 2). Autoclave with the cap loose for 30 to 40 min. Let water cool down for 24 hr. Tighten the cap only when container is at room temperature, and store in dark, cool place until use. Figure 2. 500 ml Pyrex'v glass container with a screw cap Nutrients are re-bottled in small (500-1000 ml) dark teflon or polypropylene containers.
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